Plasminogen activator inhibitor-1 is a natural inhibitor of tissue plasminogen activator. Deficiencies in plasminogen activator inhibitor-1 (PAI-1) results in delayed, or prolonged, bleeding. A high level of PAI-1 is indicative of heart disease. A reliable assay for the detection of low or high levels of PAI-1 activity is desirable.
PAI-1 has been expressed as arbitrary units (AU), wherein 1 AU of PAI-1 is the amount that inhibits 1 unit of tissue plasminogen activator (t-PA) under the conditions given. For example, in the assay as sold by Kabi the AU is measured during a 10 minute incubation of samples with standard t-PA at 37.degree. C.
A current method (Coatest.RTM., Kabi Diagnostics) for assaying PAI-1 activity in plasma or serum involves adding 40 U/ml t-PA, incubating the mixture for 10 minutes at room temperature and measuring the remaining t-PA activity in the sample. The remaining t-PA activity is converted to PAI-1 activity (AU/ml) using a standard curve which is generated by adding 40 U/ml t-PA in PAI-1 depleted plasma (0 AU/ml PAI-1) and no t-PA added (40 AU/ml PAI-1). Chmielewska et al., Clinical Chemistry, 32(3):482-485 (1986), incorporated herein by reference.
This prior art method has several drawbacks. The sensitivity or experimental error of the assay near the low and high ends of the range, in this case, 0 and 40+AU/ml (dictated by the amount of t-PA added to the sample), can be poor. For example, samples containing higher activity than 40 AU/ml PAI-1 are not detected, requiring sample dilution or increased amounts of t-PA to be added to the assay with the development of new standard curves.
PAI-1, t-PA and the complex of PAI-1.multidot.t-PA are under equilibrium in vivo, with secretion and clearance of t-PA and PAI-1, and the inhibition reaction. Since this quilibrium is not the same in a plasma sample, the prior art assay may not determine the PAI-1 activity in vivo under physiological conditions. Thus, PAI-1 activity determined in a plasma sample can be lower than that in vivo, depending upon the original t-PA activity in vivo.
This prior art assay has the further drawback in that the values produced are highly dependent upon the standard curve and the accuracy of the t-PA activity used in the assays, requiring that new standard curves be generated with new samples.
Accordingly, an assay which can be employed to accurately measure PAI-1 and t-PA activity in vivo as well as in vitro, and the half-life of t-PA activity in a sample is desirable.